Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Add 9.0 ml of distilled water to a 20 ml beaker and dissolve the first two components. While stirring, adjust the pH to 7.2 with 10N NaOH (approximately 0.7 ml). Adjust final volume to 10 ml with distilled water and filter sterilize. Solution should be used soon after preparation. Discard any unused solution.

1. Harvest cells from several cultures that are in the late logarithmic to early stationary phase of growth. Place culture vessels on ice for 10 min.

b) Add 0.8 ml of the 2.5 M Sucrose solution, remove from ice and invert until the DMSO is liquefied. Return to ice bath.

c) Add 0.2 ml of the L-Cysteine/Ascorbic Acid Solution to the DMSO solution and mix.

d) Add 6.0 ml of the CPMB-2 Basal solution and mix.

e) Add 2.0 ml HIBS and mix.

4. Resuspend the cell pellets and pool to a final volume of approximately 10 ml with the supernatant. Make a determination of the cell density and adjust the concentration of the cells between 5 x 105/ml - 1 x 106/ml using fresh medium. If the cell concentration is below 5 x 105/ml, centrifuge the cell suspension and resuspend the pellet in a volume that will yield the desired concentration.

5. After the cell concentration is adjusted, centrifuge as in step 2.

6. Remove as much supernatant as possible and determine the volume removed.

7. Resuspend the cell pellet with a volume of the cryoprotective solution equal to the volume of the supernatant removed. Invert the tube several times to obtain a uniform cell density.

13.Screw cap on tightly and incubate at a 15° horizontal slant at 35°C. Observe the culture daily and transfer when many trophozoites are observed.

Name of Depositor

LS Diamond

Special Collection

NCRR Contract

Permits

These permits may be required for shipping this product:

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.